Proc. Natl. Acad. Sci. USA Vol. 90, pp. 11094-11098, December 1993 Immunology Costimulation of T-cell activation and virus production by on activated CD4+ T cells from human immunodeficiency virus type 1-infected donors OMAR K. HAFFAR, MOLLY D. SMITHGALL, JEFFREY BRADSHAW, BILL BRADY, NITIN K. DAMLE*, AND PETER S. LINSLEY Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA 98121 Communicated by Leon E. Rosenberg, August 3, 1993 (receivedfor review April 29, 1993)

ABSTRACT Infection with the human immunodeficiency sequence (CTLA-4) (34), a structurally related to virus type 1 (HIV-1) requires T-cefl activation. Recent studies CD28 but only expressed on T cells after activation (12). have shown that interactions of the T- receptors CTLA-4 acts cooperatively with CD28 to bind B7 and deliver CD28 and CTLA-4 with their counter receptor, B7, on antigen- T-cell costimulatory signals (13). presenting cells are required for optimal T-cell activation. Here Because of the importance of the CD28/CTLA-4 and B7 we show that HIV-1 infection is associated with decreased interactions in immune responses, it is likely that these expression of CD28 and increased expression of B7 on CD4+ interactions are also important during HIV-1 infection. Stud- T-cell lines generated from seropositive donors by afloantigen ies with anti-CD28 monoclonal antibodies (mAbs) suggested stimulation. Loss of CD28 expression was not seen on CD4+ a role for CD28 in up-regulating HIV-1 long terminal repeat- T-ceU lines from seronegative donors, but up-regulation of B7 driven transcription of a reporter gene in leukemic cell lines expression was observed upon more prolonged culture. Both (14). Groux et al. (15) showed that CD28 triggering with T-ceUl proliferation and 2 mRNA accumulation in specific mAbs prevented activation-induced cell death in HlIV--infected cultures required costimulation with exogenous T-cell cultures from HIV-1-infected donors. The role ofB7 in B7 because these events were blocked by CTLA4Ig, a soluble HIV-1 infection has not been studied, but cultured cell lines form of CTLA-4 that binds B7 with high avidity. In contrast, infected with another human lymphocyte-specific retrovirus, levels ofHIV-1 RNA were not affected by CTLA4Ig, indicating human T-cell lymphotropic virus type 1 (HTLV-1), ex- that regulation of virus transcription in these cultures did not pressed the B7 protein on their surface (16). More recently, depend upon CD28-B7 engagement. Infected T cells could Freeman et al. (17) showed that several HTLV-1-transformed present alloantigen to fresh, uninfected CD4+ T cells, leading T-cell lines expressed mRNAs for B7 and CTLA-4 but not for to increased proliferation and virus spread to the activated CD28. cells. Both of these events were blocked by CTLA4Ig. Thus, We studied the expression and function of CD28/CTLA-4 chronic activation of HIV-1-infected CD4+ T cells reduces and B7 during HIV-1 infection. Specifically, we investigated expression of CD28 and increases expression of B7, thereby like infection with HTLV-1, enabling these T cells to become antigen-presenting ceOls for whether infection with HIV-1, uninfected CD4+ T cells; this might be another mechanism for was associated with increased expression of B7 on T cells, HIV-1 transmission via T-cell-T-cell contact. and if so, whether this might have functional consequences. We generated primary cultures of CD4+ T cells from HIV- 1-infected individuals by alloantigen stimulation, which pro- Infection with the human immunodeficiency virus type 1 with a (HIV-1) leads to a reduction of CD4+ T cells and a loss of vided a homogeneous population of primary T cells immune competency (1). HIV-1-infected individuals exhibit high frequency of HIV-1 infection. a marked deficiency in their ability to respond to recall (e.g., tetanus toxoid) (2). Analysis ofHIV-1-infected MATERIALS AND METHODS cells in vitro has shown defects in T-cell activation and signal transduction (3). Despite the immune defects caused by mAbs and Soluble Receptors. Origins of the mAbs used, HIV-1 infection, induction and spread of the virus requires G17-2 (anti-CD4), G10-1 (anti-CD8), 60.1 (anti-CD11b), 63D3 T-cell activation (4-6). Thus, HIV-1 infection is intimately (anti-CD14), FC2 (anti-CD16), iF5 (anti-CD20), HB1Oa (anti- involved with function of the immune system. MHCII), and 9.3 (anti-CD28) have been presented (11, 18). Optimal antigenic stimulation of CD4+ T cells requires (i) mAb 11D4 (anti-CTLA-4) has been described (13). Anti-CD3 engagement ofthe T-cell receptor-CD3 complex by antigenic mAb Leu-4 conjugated to peridinin chlorophyll protein peptides presented in the context ofmajor histocompatibility (PerCP) and anti-B7 mAb BB1 were purchased from Becton complex (MHC) molecules, and (ii) additional costimulatory Dickinson. Anti-HIV-1 mAb 25-3 was reactive with the signals provided by engagement of accessory receptors on HIV-1 core protein p24 (Genetic Systems, Seattle). Human the T-cell surface with counter receptors on antigen- CTLA4Ig (the soluble form of CTLA-4 that binds B7) has presenting cells (for review, see ref. 7). In addition to been described (12). lymphocyte function-associated antigen 1 (LFA-1) and CD2 (for review, see ref. 7), recent work has elucidated the Abbreviations: HIV-1, human immunodeficiency virus type 1; importance ofthe CD28 receptor in T-cell-dependent immune HTLV-1, human T-cell lymphotropic virus type 1; mAb, monoclonal responses (8, 9). A counter receptor for CD28 is the B7 antibody; IL-2, ; CTLA-4, cytolytic T-lymphocyte- molecule expressed on activated antigen-presenting cells (10, associated sequence; FITC, fluorescein isothiocyanate; CTLA4Ig, 11). B7 also binds to cytolytic T-lymphocyte-associated soluble form ofCTLA-4 that binds B7; LFA-1, lymphocyte function- associated antigen 1; MLC, mixed lymphocyte cultures; MHC, major histocompatibility complex; EBV-LCL, Epstein-Barr virus- The publication costs of this article were defrayed in part by page charge transformed (s). payment. This article must therefore be hereby marked "advertisement" *Present address: Wyeth-Ayerst Research, CN 8000, Princeton, NJ in accordance with 18 U.S.C. §1734 solely to indicate this fact. 08543-8000. 11094 Downloaded by guest on October 1, 2021 Immunology: Haffar et al. Proc. Natl. Acad. Sci. USA 90 (1993) 11095 Generation of Alloantigen-Primed T-Cell Lines. CD4+ T A CD2 CD28 LFA-1 MHC Class 11 were isolated from peripheral blood mononu- clear cells from HIV-1-seronegative and -seropositive do- 806 nors, as described (18). Purified CD4+ T cells (>90% pure) were cocultured with irradiated (10,000 rads; 1 rad = 0.01 Gy) Epstein-Barr virus-transformed B cells (EBV-LCL) from a Z29 E; seronegative donor at weekly intervals. After the initial stimulation, the ratio ofstimulators to responders was altered from 1:2 to 1:5 for all subsequent stimulations, and culture Z39 Z L = medium (RPMI 1640 medium/10%o human serum) was sup- plemented with human interleukin 2 (IL-2) at 10 units/ml. Expanded T cells were collected and depleted of CD8+ T Z44 0 cells by complement lysis after mAb G10-1 binding. 1 2 0 30 1 30 1 2 3 Cocultivation and Mixed Lymphocyte Response Assays. Log Fluorescence Intensity Irradiated (10,000 rads) infected alloreactive T cells were used as stimulator cells in mixed lymphocyte cultures (MLC) B with freshly isolated uninfected CD4+ T cells. The responder cells were derived from the same seronegative donor as the Z64 CD3 CD4 CD28 B7 B cells used in the alloactivation of the HIV-i-infected cells. 2Wk A A The MLC were maintained in culture medium with or without CTLA4Ig (10 ,g/ml). Cell proliferation was measured by 3 Wk -AA l [3H]thymidine incorporation, and HIV-1 production was measured by evaluating the p24 concentrations in the culture Z39 medium using specific enzyme immunoassay (5). ,-II 77A7/\ I\ L 11/1- Immunostaining of Cell Surface-Associated . Cell- .. surface expression of proteins was evaluated by multicolor 3Wk E -I immunofluorescent staining. After staining, cells were z washed with phosphate-buffered saline and fixed overnight 4Wk wD[X L l with 2% paraformaldehyde solution, pH 7.2, at 40C. Intact cells were analyzed by using either an EPICS V (Coulter) or 400 FACScan (Becton Dickinson) instrument. 2 Wk A 1] A 11A I RNA Blot Analysis. RNA was prepared from the alloreac- tive cell lines at various times during the fourth round of 3 Wk A -Al stimulation, fractionated by electrophoresis, and transferred to nitrocellulose filters as described (9). The filters were probed for various RNAs as described (9, 13). The HIV probe 1 2 3 1 2 3 1 2 3 1 2 3 was a 3.1-kb DNA fragment excised from the gag open Log Fluorescence Intensity reading frame by digestion of the plasmid pMgtre (provided with BssHII and Xba FIG. 1. (A) Cell-surface expression of specific receptors in HIV- by Alan Senear, Bristol-Myers Squibb) 1-infected and -uninfected alloantigen-primed CD4+ T-cell lines. I. Cell-surface expression of accessory molecules CD2, CD28, and PCR Analysis. Presence of B7-specific message was eval- LFA-1, as well as MHC class II protein, was evaluated in HIV-1- uated by PCR of cDNA generated from reverse transcription infected (Z29, Z39, and Z44) and uninfected (806) lines at day 7 ofthe of cellular RNA fractions (19). The primers used were as fourth round of stimulation using fluorescein isothiocyanate (FITC)- follows: for B7, 5'-CTGAGCTCTATGCTGTTAGC-3' conjugated mAbs. (B) Kinetic analysis of down-regulation of CD28 (sense) and 5'-ACAGAATTCTGCGGACACTGTTATA- and up-regulation of the counter receptor B7. Cells from infected CAGG-3' (antisense); for CD19, 5'-CTGAACCAGAGCCT- (Z29 and Z64) and uninfected (400) lines were assayed for expression CAGCCAG-3' (sense) and 5'-TGCTCGGGTTTCCATAA- of CD28 and B7 in parallel with CD3 and CD4 at day 7 of various GACG-3' (antisense); for hypoxanthine phosphoribosyl- rounds of stimulation, as indicated using FITC-conjugated mAbs. transferase 5'-GTTGGATACAGGCCAGACTTTGTTG-3' CTLA4Ig conjugated to FITC was used for B7-specific staining. (sense), and 5'-GATTCAACTTGCGCTCATCTTAGGC-3' altered in either the seropositive lines Z29, Z39, and Z44 or (antisense). the seronegative line 806 (Fig. 1A). Also presented in Fig. 1A is the relative expression of MHC class II molecules on the RESULTS activated T cells from the various donors. Fig. 1B shows that reduction in cell-surface expression of HIV Infection Specifically Alters the Expression ofthe T-Cell and Costimulatory Receptor CD28. Primary T-cell lines were CD28 in HIV-1-infected cultures was gradual paralleled generated by alloantigen stimulation ofCD4+ T lymphocytes the down-modulation ofCD4 cell-surface reactivity (compare purified from HIV-1-seropositive (Z29, Z36, Z39, Z44, and lines Z64 and Z39 with line 400). In contrast, cell-surface Z64) and seronegative (400, 742, 806, 851, and 948) donors. expression of CD3 was not affected. Loss of CD4 expression Proliferation of the lines required the addition of antigen- in HIV-1-infected cultures was shown to correlate with presenting cells. Cell-surface expression of various T-cell expression of viral proteins (20-23). Analysis of the alloac- activation-associated receptors was evaluated using specific tivated cultures showed increased virus production, and mAbs. Fig. 1A shows that surface reactivity ofcells from the virus spread with increased periods of stimulation (data not seropositive donors Z29, Z39, and Z44 with anti-CD28 mAb shown). Levels of infection at round four of stimulation was significantly reduced compared with the surface reac- varied between lines, but HIV-specific immunofluorescent tivity of cells from the seronegative donor 806 when assayed assays revealed that levels of infection as high as 50-90%o after 4 weeks of allostimulation. This decrease in CD28 could be achieved for some cell lines. reactivity was evident in T-cell lines from the five seropos- Expression of CD28 and its homologue CTLA-4 were itive donors (Fig. 1 A and B and data not shown). In contrast, evaluated in cells from donors Z29 and 948 during the fifth cell-surface reactivity with mAb to CD2 and LFA-1 were not round of stimulation. Fig. 2 shows that the CD4+ CTLA-4+ Downloaded by guest on October 1, 2021 11096 Immunology: Haffar et al. Proc. Natl. Acad. Sci. USA 90 (1993) DAY 2 DAY 7 A A B C D E SIze ( bp) 1018 - 506/5i77396: -CD19 A 344- 4-B7 298 - 280 -* HPRT I 20 1' 154 134 c- I- - H 0- B Z29 806 3 WK 3 Wk Ln 0 0 ~~0 7~39 3Wk 0 -j .S~c ~~ \ I L.1' 1L 2 744 851 3 W k 7 Wk 0 C0 0 82 3 .og Fluorescence Intensity

CD4 o FIG. 3. (A) B7 mRNA is present in infected and uninfected lines. Log Fluorescence Intensity cDNA was generated by reverse transcription of RNA derived from infected (Z36, lane C, and Z44, lane D) and uninfected (400, lane B) FIG. 2. Kinetic analysis of CD28 and CTLA-4 expression in cell lines after the fourth round of stimulation. B7 sequences were primed T-cell lines. Three-color fluorescence was used to evaluate amplified by PCR, fractionated in agarose gels and visualized with expression ofCD28 and CTLA-4 at days 2 and 7 during the fifth round ethidium bromide staining. RNA fractions from the EBV-LCL (lane of stimulation of infected (Z29) and uninfected (806) lines. The A) and Jurkat cells (lane E) were used as positive and negative anti-CD3 mAb Leu-4 was conjugated to peridinin chlorophyll protein controls, respectively, for B7 mRNA expression. PCR analysis for (PerCP), the anti-CD4 mAb G17-2 was conjugated to FITC, the CD19 mRNA (lane A) served as an internal control to confirm the anti-CD28 mAb 9.3 and the anti-CTLA-4 mAb l1D4 were conjugated absence of RNA from residual B cells in our T-cell fractions (lanes to biotin. After binding of mAbs, cells were incubated with strepta- B-D). Evaluation of hypoxanthine phosphoribosyltransferase vidin-phycoerythrin. Costaining ofCD3+ cells for CD4 and CD28 or (HPRT) RNA confirmed equal loading of samples. (B) Relative CTLA-4 was evaluated. expression ofB7 on infected (Z29, Z39, and Z44) and uninfected (806 and 851) lines was evaluated after three rounds (3 Wk) and seven population represented 60%o of the Z29 culture but only 28% rounds (7 Wk) of stimulation using CTLA4Ig conjugated to FITC. of the 948 culture when evaluated 2 days after activation. At day 7 after activation, cell-surface reactivity with anti- positive cells (806 and 851 vs. Z29, Z39, and Z44, respec- CTLA-4 mAb returned to basal levels in both cultures. Two tively). The B7 transcript was not derived from residual B additional HIV-infected lines also showed enhanced expres- cells because CD19 mRNA was not detected (Fig. 3A, lane A sion of CTLA-4 (data not shown). In contrast, at day 2 vs. lanes B, C, and D). postactivation the CD4+ CD28+ populations were compara- Function of CD28/CTLA-4 Costimulatory Molecules on ble in both the Z29 and 948 cultures (81% and 95%, respec- HIV-1-Infected Lines. Costimulation of T-cell activation via tively). However, at day 7 after activation CD28 expression the CD28 receptor results in accumulation ofIL-2 mRNA and was reduced in the Z29 culture (CD4+ CD28+ = 58%) and proliferation (9). Therefore, the effect of HIV-1 infection on correlated with loss ofCD4 expression because CD4+ CD28- activation of the alloantigen-primed T-cell lines was evalu- cells were not detected (see lower right quadrant). Expres- ated by assaying for IL-2 transcripts. Fig. 4 shows that the sion of CD28 in the 948 culture remained relatively stable. HIV-1-infected and -uninfected lines expressed equivalent Expression of CD28 Counter Receptor B7 on Activated T levels of IL-2 mRNA. This response was inhibited in all cell Cells. Infection of human T cells with HTLV-1 up-regulated lines equally by addition of CTLA4Ig to the cultures. Thus, B7 expression (16). Because HIV-1 and HTLV-1 are both regulation of IL-2 production by the CD28/CTLA-4 recep- retroviruses, we evaluated whether infection of T cells with tors was not greatly affected by HIV-1 infection in these HIV-1 also induces B7 expression. Fig. 1B shows that primary T-cell cultures. The transient reduction in CD28 cell-surface reactivity with mAb to B7 was higher in the mRNA levels early after activation (see 6-hr time point) was HIV-infected lines Z64 and Z39 compared with uninfected recently reported (24) and was not observed for CTLA-4 line 400. Moreover, the increase in B7 expression evident in mRNA (Fig. 4). However, the expression of CTLA-4 mRNA the HIV-infected lines was gradual over several weeks of but not CD28 mRNA was blocked by CTLA4Ig when as- allostimulation (see donors Z64 and Z39). PCR analysis sayed at both 6-hr and 24-hr postactivation. showed the presence of a B7-specific transcript in RNA Normal induction and regulation ofIL-2 mRNA in infected fractions from infected and uninfected lines (Fig. 3A). How- and uninfected cultures was consistent with the presence of ever, as shown in Fig. 3B, several additional weeks of relatively equivalent levels of CD28 and CD28 mRNA early allostimulation were required to achieve levels of B7 expres- in the stimulation cycle (donor Z29; Fig. 2, day 2, and Fig. 4, sion on the seronegative cells comparable to those on sero- respectively, as compared with seronegative lines). In addi- Downloaded by guest on October 1, 2021 Immunology: Haffar et al. Proc. Natl. Acad. Sci. USA 90 (1993) 11097 806 Z29 13c `44 cell-adsorbed virions. Therefore, the levels ofHIV-1 RNA in -. the alloantigen-primed T-cell lines were not regulated by !mRle r. 2 r '- CD28-B7 association. --tT-r +--- Role of B7 Protein in T-Cell Activation and Virus Spread. The observation that HIV-1-infected cultures expressed both IL-22 MHC class II (see Fig. 1B) and B7 suggested that these T-cell lines may be capable of presenting alloantigen to fresh CD4+ 2,,,,8S; , T cells. This question was addressed by testing the ability of CD28 . :.8S the HIV-1-infected lines to activate CD4+ T cells in MLC. '9 s CD4+ T cells were prepared from a seronegative donor (see CnTLA-4 _ Materials and Methods) and cocultured with irradiated sero- positive stimulator cells for 7 days. Fig. 5 Upper shows that HV I# Wbi *I* 28S the responder cells proliferated as determined by incorpo- ration of [3H]thymidine. Magnitude ofthe response depended on the stimulator/responder cell ratios (see abscissa). In GAPDH ------___- _,,"1'8£ addition, virus production was amplified in these cocultures, indicating spread to the responder cells (Lower). Increased FIG. 4. Northern blot analysis of mRNA e:xpression after acti- virus production required the addition of fresh cells because vation of the primed T-cell lines. Cells from infe.cted and uninfected cultures containing only irradiated stimulator cells, carried in cell lines (as indicated) were activated by additLion of EBV-LCL in parallel, yielded basal levels of virus (=1-2 ng of p24 per ml the presence or absence of CTLA4Ig at 10 pg/rml (as indicated). At at the highest input of cells; data not shown). Virus spread 6 and 24 hr after activation RNA was extracte4d from the cells and further confirms the activated state of the responder cells, analyzed by Northern blot for expression of sjpecific mRNA using because HIV-1 cannot productively infect resting cells (25, 32P-radiolabeled probes. Equal loading of material was ensured by 26). Fig. 5 also shows that both the proliferative response and evaluating GAPDH mRNA levels. virus production (Upper and Lower, respectively) were in- hibited by the presence of that both tion, the enhanced expression of CTLA-4Arin4inHIV-iHIV*Infectednected processes were costimulatedCTLA4Ig,by the B7suggestingmolecule. lines (Fig. 2 and data not shown) may conitribute to normal The concentrations ofCTLA4Ig resulting in 50% inhibition activation of the T cells. (IC50) of proliferation and virus production were 0.31 pg/ml Although HIV-1 induction depends on T-cell activation and 0.08 pg/ml, respectively, as determined in cultures using (4-6), Fig. 4 shows that after a transient reduction at 6-hr EBV-LCL stimulation of HIV-1-infected cells (Z64; data not postactivation, the expression and/or accuimnulation ofHIV-i shown). genomic RNA (Z29, Z39, and Z44) were not altered in the presence of CTLA4Ig by 24-hr postacti'vation. The viral DISCUSSION transcripts detected in these experimentts represent cell- We used allostimulated primary CD4+ T-cell lines to inves- associated RNA that could encompass RINA derived from tigate the effect of HIV-1 infection on the expression of z36 Z44 .0 50

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FIG. 5. B7 on HIV-infected T cells costimulates proliferation and virus production in MLC. Cells from Z36 and Z44 HIV-infected cultures were irradiated and used as stimulator cells in MLC with freshly isolated uninfected CD4+ T cells. The cultures contained various stimulator/responder cell ratios, as indicated. Proliferation of the responder cells was evaluated by measuring [3H]thymidine incorporation by cells (cpm x 10-3, Upper) on day 3 ofcoculture, and virus production was evaluated by measuring HIV-1 p24 levels (ng/ml, Lower) in the culture supernatants on day 7 of coculture. The role of CD28-B7 in mediating these events was evaluated by assaying the effect of CTLA4Ig (10 ,g/ml) (open symbols) in the culture. Downloaded by guest on October 1, 2021 11098 Immunology: Haffar et al. Proc. Natl. Acad. Sci. USA 90 (1993) specific T-cell costimulatory receptors and on T-cell func- We thank Drs. Steve Kanner and Jeff Ledbetter for their critical tion. Analysis of virus production by p24-specific enzyme review of the manuscript. We also thank Joanne Greene and Kerry immunoassay and antigen-specific immunofluorescence as- Klussman for technical assistance. say showed that virus spreads in the cultures with repeated 1. Lane, H. C., Depper, J. M., Greene, W. C., Whalen, G., Wald- stimulation (data not shown). By week 4 of stimulation high mann, T. A. & Fauci, A. S. (1985) N. Engl. J. Med. 313, 79-84. infection levels (50-90%) can be achieved with this system. 2. Gurley, R. J., Ikeuchi, K., Byrn, R. A., Anderson, K. & Groop- In addition, cells in the alloreactive lines described here can man, J. E. (1989) Proc. Natl. Acad. Sci. USA 86, 1993-1997. be uniformly activated to produce HIV-1 by addition of the 3. Linette, G. P., Hartzman, R. J., Ledbetter, J. A. & June, C. H. EBV-LCL. These properties rendered the system very well (1988) Science 241, 573-576. 4. Diegel, M. L., Moran, P. A., Gilliland, L. K., Damle, N. K., suited to addressing specific questions regarding HIV-1- Hayden, M. S., Zarling, J. M. & Ledbetter, J. A. (1993) AIDS Res. CD4+ T-cell interactions. Human Retroviruses 9, 465-473. Our results indicate that HIV-1 modulates the expression 5. Haffar, 0. K., Moran, P. A., Smithgall, M. D., Diegel, M. L., of specific cell-surface proteins. Several reports have ad- Sridhar, P., Ledbetter, J. A., Zarling, J. M. & Hu, S.-L. (1992) J. Virol. 66, 4279-4287. dressed the reduced expression of CD4 in the presence of 6. Moran, P. D., Diegel, M. L., Sias, J. C., Ledbetter, J. A. & HIV-1 (20-23). We showed that infection with HIV-1 re- Zarling, J. M. (1993) AIDS Res. Human Retroviruses 9, 455 464. sulted in the down-modulation of CD28 but not other acces- 7. Schwartz, R. H. (1990) Science 248, 1349-1356. sory molecules, such as CD2 or LFA-1. Kinetic analysis 8. Azuma, M., Cayabyab, M., Buck, D., Phillips, J. H. & Lanier, during one cycle of stimulation suggested that down- L. L. (1992) J. Exp. Med. 175, 353-360. 9. Linsley, P. S., Brady, W., Grosmaire, L., Aruffo, A., Damle, N. K. regulation of CD28 expression occurred late in the cycle and & Ledbetter, J. A. (1991) J. Exp. Med. 173, 721-730. most likely reflected expression or accumulation of viral 10. Freeman, G. J., Gray, G. S., Gimmi, C. D., Lombard, D. B., Zhou, proteins. The gradual reduction in CD28 expression in the L.-J., White, M., Fingeroth, J. D., Gribben, J. G. & Nadler, L. M. cell cultures over several rounds of stimulation correlated (1991) J. Exp. Med. 174, 625-631. with virus spread cells. 11. Linsley, P. S., Clark, E. A. & Ledbetter, J. A. (1990) Proc. Natl. and increased number of infected Acad. Sci. USA 87, 5031-5035. Expression of CTLA-4 was enhanced in several HIV-1- 12. Linsley, P. S., Brady, W., Urnes, M., Grosmaire, L. S., Damle, infected lines compared with the seronegative line 948 (Fig. N. K. & Ledbetter, J. A. (1991) J. Exp. Med. 174, 561-569. 2 and data not shown). Because CTLA-4 is the high-affinity 13. Linsley, P. S., Greene, J. L., Tan, P., Bradshaw, J., Ledbetter, receptor for B7 (12), this enhanced expression may have J. A., Anasetti, C. & Damle, N. K. (1992) J. Exp. Med. 176, 1595-1604. compensated functionally for the down-regulation of CD28. 14. Tong-Starkesen, S. E., Luciw, P. A. & Peterlin, B. M. (1989) J. Activation of the T-cell lines, as measured by induction of Immunol. 142, 702-707. IL-2 mRNA and proliferation (data not shown), was not 15. Groux, H., Torpier, G., Monte, D., Mouton, Y., Carpon, A. & affected by the presence of HIV-1. Although stimulation of Ameisen, J.-C. (1992) J. Exp. Med. 175, 331-340. CD28 was shown to activate the HIV long terminal repeat- 16. Valle, A. P., Garrone, H. Y., Bonnefoy, J.-Y., Freedman, A. S., driven of a our data show Freeman, G., Nadler, L. M. & Banchereau, J. (1990) Immunology transcription reporter gene (14), 69, 531-535. that early accumulation ofHIV-1 genomic RNA in the primed 17. Freeman, G. J., Lombard, D. B., Gimmi, C. D., Brod, S. A., Lee, T-cell lines was not dependent on CD28-B7 association K., Laning, J. C., Hafler, D. A., Dorf, M. E., Gray, G. S., Reiser, because CTLA4Ig could not block the RNA responses to H., June, C. H., Thompson, C. B. & Nadler, L. M. (1992) J. alloantigen. Immunol. 149, 3795-3801. We also showed that HIV-1 accelerates B7 expression in 18. Damle, N. K., Klussman, K. & Aruffo, A. (1992) J. Immunol. 148, was 665-671. alloactivated T-cell lines. While this work being prepared 19. Symington, F. W., Brady, W. & Linsley, P. S. (1993) J. Immunol. for publication, two reports presented the expression of B7 150, 1286-1295. on CD4+ T cells after prolonged activation in vitro (27, 28). 20. Garcia, J. V., Alfano, J. & Miller, D. (1993) J. Virol. 67, 1511-1516. The mechanism for induction of B7 in T cells is not known; 21. Hoxie, J. A., Alpers, J. D., Rackowski, J. 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P. & Lamonica, activation of the responder cells and virus spread in the C. A. (1990) EMBO J. 9, 1551-1560. culture, suggesting that the B7-CD28 interaction could be 26. Zack, J. A., Arrigo, J. J., Weitsman, S. R., Go, A. S., Haislip, A. important for virus transmission from stimulator to responder & Chen, I. S. Y. (1990) Cell 61, 213-222. T cells. Thus, the B7-CD28 adhesion system may facilitate 27. Azuma, M., Yssel, H., Phillips, J. H., Spits, H. & Lanier, L. (1993) virus spread byjuxtapositioning of the T cells, as was shown J. Exp. Med. 177, 845-850. 28. Sansom, D. M. & Hall, N. D. (1993) Eur. J. Immunol. 23, 295-298. for the LFA-1-intercellular adhesion molecule 1 system (33). 29. Valle, A., Aubry, J. P., Durand, I. & Banchereau, J. (1991) Int. Because HIV-1 does not productively infect resting T cells Immunol. 3, 229-235. (25, 26), previous analysis of virus transmission in T-cell 30. Nabavi, N., Freeman, G. J., Gault, A., Godfrey, D., Nadler, L. 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